PROJECT ABSTRACT The long-term objective of this application is to develop a detailed understanding of the molecular and structural basis for red cell membrane function in health and disease. The aim of this application is to define the origins of red cell membrane material behavior at the molecular level by combining structural and functional information on individual proteins with detailed biophysical characterization of red cells. To achieve our stated objective we have designed a series of studies with the following specific aims: 1) Explore the hypothesis that distinct macromolecular complexes of membrane proteins are assembled in the red cell membrane and linkage of these complexes to the membrane skeleton regulates several membrane functions. 2) Explore the hypothesis that dynamic regulations of lateral linkages between skeletal proteins play a key role in modulating membrane mechanical stability. In particular, we will explore the dynamic regulation of linkage between spectrin dimers and between junctional complex proteins, spectrin, 4.1R, actin and adducin. 3) Explore the hypothesis that interaction of lipids with skeleton modulates membrane function. 4) Explore the hypothesis that significant structural reorganization of integral and skeletal proteins is responsible for the documented changes in membrane material properties during maturation of reticulocytes into mature red cells. For the proposed studies we will employ a variety of biophysical strategies including fluorescence-imaged micropipet aspiration, the technique of magnetic tweezers and ektacytometry. Human red cells with defined protein defects and red cells from knockout and knockin mice will be used to critically evaluate the contribution of various proteins to membrane function. We anticipate that successful execution of our proposed studies will enable us to critically test the various hypotheses for the origins of membrane material behavior at the molecular level and offer significant insights into functional consequences of altered membrane organization in pathologic red cells. We anticipate that findings from these studies will lead to development of better clinical management strategies for inherited and acquired red cell disorders which affect over a billion humans around the world. Project Narrative: Inherited and acquired red cell disorders including hereditary spherocytosis, hereditary elliptocytosis, hereditary ovalocytosis, thalassemias, sickle cell disease, enzymopathies, malarial anemia and iron deficiency anemia affect over billion humans. A feature of all these disorders is altered membrane function which in turn contributes to clinical manifestations. The research project will explore the molecular and structural basis for red cell membrane material behavior in health and disease. It is anticipated that the findings from the proposed studies will contribute to better clinical management of red cell disorders and for developing new therapeutic approaches for management of hemolytic anemias.